The physiology of many organs in mammals is regulated by hormones. These hormones include steroid hormones, thyroid hormones, metabolites of vitamins, such as all trans retinoic acid, 9-cis retinoic acid, vitamin D and its metabolite 1,25 dihydroxyvitamin D3. These hormones bind to intracellular receptors which regulate expression of genes (O""Malley, 1990).
There are a variety of receptors which respond to hormones. Osteoblasts and osteoclasts respond to steroid hormones, vitamin D and retinoic acid. Mammary epithelial cells and breast carcinoma cells respond to estrogens, progesterone, retinoic acid and glucocorticoids. Lymphocytes respond to glucocorticoids.
The response of receptors to hormones is particularly important in the development of a number of diseases, including cancer, osteoporosis and chronic inflammatory disease. For example, the vitamin D receptor is strongly implicated in the evolution of osteoporosis (Morrison, 1994).
The hormone receptor family is called the nuclear hormone receptor family and consists not only of receptors whose ligands are known, but also of an increasing number of orphan receptors whose ligands are unknown (O""Malley, 1990).
The nuclear hormone receptors can be divided into several domains which include the hormone (ligand) binding domain, the DNA-binding domain and the transactivation domain (O""Malley, 1990). The DNA-binding domain consists of two zinc fingers and is responsible for the receptor""s binding to the DNA response elements which are found in the promoter and enhancer regions of the genes whose expression are regulated by these receptors. Once a hormone binds to its receptor, the receptor binds to the DNA thereby inducing gene transcription.
Proteins which modulate hormone receptor induced gene transcription are poorly understood. Such proteins are present in the nucleus of the cell and inhibit or promote the binding of a hormone to its receptor.
To help design pharmaceuticals and therapies for certain diseases, one must understand the function of certain intracellular proteins and their role in modulating hormone responsiveness. Isolation and purification of these proteins would help in assessing whether they inhibit or promote hormone receptor induced gene transcription. Once such proteins are isolated, manipulation of such proteins could further inhibit or promote hormone receptor induced gene transcription. Synthetic peptides which bind to such proteins could be used to promote hormone receptor induced gene transcription. Pharmaceuticals including such peptides or their mimetics could be used to inhibit hormone receptor induced gene transcription. Gene therapy could be used to inhibit or promote hormone receptor induced gene transcription.
A need exists to identify amino acid sequences that are conserved in hormone receptors, so that particular peptides and proteins may be designed and used in modulating hormone responsiveness. This would lead to improved methods of treating a variety of diseases, disorders and abnormal physical states in mammals by regulating hormone receptor induced gene transcription in mammalian cells.
Calreticulin was initially identified as the major Ca2+-storage protein in the sarcoplasmic reticulum of skeletal muscle (Ostwald and MacLennan, 1974). Subsequent work has revealed that the protein can also be detected in the endoplasmic reticulum of non-muscle tissues (Fliegel et al., 1989a; Opas et al., 1991). Calreticulin has been considered to be a resident protein of the endoplasmic reticulum of a cell, where it is thought to behave as a calcium binding protein due to its high capacity calcium binding properties (Michalak et al., 1992). Calreticulin possesses many diverse functional domains such as high affinity, low capacity- and low affinity, high capacity-Ca2+-binding sites, a C-terminal KDEL endoplasmic reticulum retention signal, and a nuclear localization signal (Michalak et al., 1992).
It has been suspected that calreticulin is also present in the nucleus of a cell (Opas et al., 1991), and it has been shown to have a consensus nuclear localization sequence (Michalak, 1992) which is highly homologous to that of histone proteins. However, before this invention, its presence in the nucleus was unconfirmed and its function in the nucleus was unknown.
This invention relates to isolated and purified proteins, such as calreticulin and mimetics of calreticulin, for a novel use of modulating hormone responsiveness. These proteins are useful in gene therapy and in manufacturing pharmaceuticals for treating a variety of diseases, including cancer, osteoporosis and chronic inflammatory disease. The proteins bind to an amino acid sequence KXFFX1R, wherein X is either G, A or V and X1 is either K or R [SEQ ID NO:1]. This sequence is present in the DNA-binding domain, and is critical for the DNA binding activity of a variety of hormone receptors, including glucocorticoid receptor, mineralcorticoid receptor, androgen receptor, progesterone receptor, estrogen receptor, retinoic acid receptor, thyroid hormone receptor and vitamin D receptor. Proteins which bind to this sequence inhibit hormone receptor induced gene transcription. The invention includes isolated DNA molecules for these proteins, methods of treating diseases using these proteins, synthetic peptides and their mimetics, and kits containing these proteins, synthetic peptides or their mimetics.
This invention relates to a peptide and peptide mimetic having the amino acid sequence [SEQ ID NO:1] KXFFX1R, wherein X is either G, A or V and X1 is either K or R which promotes hormone receptor induced gene transcription. The invention includes isolated DNA molecules for these peptides, methods of treating diseases using these peptides, synthetic peptides and their mimetics, and kits containing these proteins, synthetic peptides or their mimetics. Preferably the peptides or peptide mimetics are from 6 to 100 amino acids.
This invention relates to a product for modulating hormone responsiveness. Preferably such a product is calreticulin which inhibits hormone receptor induced gene transcription. More preferably, the product is the N-domain of calreticulin. In another case, the product is a mimetic of calreticulin. The product binds to the amino acid sequence [SEQ ID NO:1] KXFFX1R, wherein X is G, A or V and wherein X1 is K or R.
In another case, the product for modulating hormone responsiveness is an antibody to calreticulin or a short peptide which binds to calreticulin. Such an antibody or peptide could promote hormone induced gene transcription by inhibiting calreticulin-hormone receptor interactions.
This invention relates to a peptide or peptide mimetic selected from a group [SEQ ID NOS:2-6] consisting of: KGFFRR, KVFFKR, KAFFKR, KGFFKR, TGFFKR or modified derivatives of these peptides. Preferably the peptide is from 6 to 100 amino acids.
This invention also relates to a peptide or peptide mimetic which reverses selectively calreticulin inhibitions of receptor binding to DNA response elements is part of this invention. One peptide which reverses selectively calreticulin inhibitions of retinoic acid to its DNA response elements is KLDFFKR [SEQ ID NO:45]. Another peptide which reverses selectively calreticulin inhibitions of androgen receptor binding to its DNA response elements is a peptide comprising a sequence selected from a group consisting of GLGFFKR [SEQ ID NO: 44], KLGFFGR [SEQ ID NO:48] and KLGFFKG [SEQ ID NO:49]. Preferably these peptides are from 6 to 100 amino acids.
The invention described in this patent application includes an isolated DNA molecule encoding an amino acid sequence for use in modulating hormone responsiveness. The isolated DNA molecule may encode the amino acid sequence for calreticulin. It may encode the amino acid sequence for part of a mimetic of calreticulin. It may encode a first amino acid sequence that binds to a second amino acid sequence [SEQ ID NO:1] KXFFX1R, wherein X is G, A or V and wherein X1 is K or R.
The invention described in this patent application includes a method of treating a disease, disorder or abnormal physical state in a mammal by regulating hormone receptor induced gene transcription in a cell. The method could include regulating the activity, quantity or stability of a peptide or peptide mimetic of the present invention for use in hormone receptor induced gene transcription. The peptide could be one that includes or binds to the amino acid sequence [SEQ ID NO:1] KXFFX1R, wherein X is G, A or V and wherein X1 is K or R. One protein which binds to such sequence is calreticulin. The hormone receptor could be one selected from a group consisting of: glucocorticoid receptor, mineral corticoid receptor, androgen receptor, progesterone receptor, estrogen receptor, retinoic acid receptor, thyroid hormone receptor, vitamin D receptor and orphan receptors. The disease or disorder could be one selected from a group consisting of breast cancer, prostate cancer, promyelocytic leukemia, solid tumors, chronic inflammatory disease, such as arthritis and osteoporosis.
The method of treating the disease could include administering to the mammal a pharmaceutical comprising a peptide, or a peptide mimetic of the invention and a pharmaceutical carrier. Another method of treating the disease could include administering to the mammal a pharmaceutical comprising an inhibitor of the protein and a carrier. A suitable carrier could be a lipid vesicle. As an alternative, the method could include decreasing or eliminating the quantity of calreticulin present in the cell; or decreasing the stability of calreticulin present in a cell.
In another embodiment, the invention is an isolated and purified peptide with the amino acid sequence KGX1X2X3R where one or more of X1, X2 or X3 is a basic amino acid [SEQ ID NO:67], and the peptide binds to calreticulin. In a preferred embodiment X1, X2 or X3 are selected from the group consisting of lysine, arginine, histidine, phenylalanine and tyrosine. More preferably, at least one of X1, X2 or X3 is arginine. The peptide can be one of the group consisting of KGRFKR [SEQ ID NO:52], KGFRKR [SEQ ID NO:53], KGRFRR [SEQ ID NO:58] and KGFRRR [SEQ ID NO:59]. Preferably, the peptide is from about 6 to 100 amino acids. The invention also includes a pharmaceutical composition comprising the peptide and a pharmaceutically acceptable carrier. In a preferred embodiment, the carrier is a lipid vesicle.
In another embodiment, the invention is an isolated and purified peptide comprising an amino acid sequence selected from the group consisting of KV(N-acetylated)AFKR [SEQ ID NO:63], KVFFKR [SEQ ID NO:3], KGFFKR [SEQ ID NO:5], KAFFKR [SEQ ID NO:4], GGFFRR [SEQ ID NO:56], KVFFKR (all D-amino acids), KGFFKR (all D-amino acids), KGFFRR [SEQ ID NO:2], GGFFKR [SEQ ID NO:50], KGFFRG [SEQ ID NO:60], AVFFKR [SEQ ID NO:62], KVFFAR [SEQ ID NO:64] and KVAFKR [SEQ ID NO:63], where the peptide binds calreticulin. Preferably, the peptide is from about 6 to 100 amino acids. The invention also includes a pharmaceutical composition comprising the peptide and a pharmaceutically acceptable carrier. In a preferred embodiment, the carrier is a lipid vesicle.
The invention also includes a method of medical treatment of a patient having cancer, chronic inflammatory disease, osteoporosis or other bone disorders comprising administering using the pharmaceutical compositions described herein.